1. Field of the Invention
In many assays, there is an interest in determining the amount of rate of change in the concentration of NADH. A large number of enzymes of interest use NAD or NADP or the reduced forms as coenzymes. The rate of formation of the reduced form can be followed by the fluorescence of the reduced form. However, in most situations, a significant amount of endogenous fluorescence is present at the wavelength of the NADH emission. In view of the low quantum yield of fluorescence of NADH, small concentrations of the reduced coenzyme is quantiated by fluorescence spectroscopy only marginally more effectively than by adsorption spectroscopy.
It is therefore desirable to find ways to enhance the signal obtained from the NADH. Desirably, the ways should be simple and not subject to interference, particularly in view of the relatively large amounts of NAD derivatives which will usually be encountered in the assay medium.
2. Description of the Prior Art
Holbrook and Wolfe, Biocheminstry (1972), 41, 2499, describes fluorescent studies of NADH bound to malate dehydrogenase. Torikata et al., J. Biol. Chem. 254, 3516-3520 (1979) describes NAD quenching of tryptophan fluorescence in malate dehydrogenase. Baici et al., Eur. J. Biochem., 83, 601-607 (1978) describes the fluorescent properties of sNADH and its complex with octopine dehydrogenase. U.K. Pat. No. 1,532,694 teaches using NADH fluorescence in enzyme assays.